JP2010540488A - Glycoproteins and glycosylated cells and methods for their preparation - Google Patents

Abstract

The present invention not only relates to glycosyl carbamoylation methods relevant to the preparation of novel glycoproteins and glycopeptides (especially glycoproteins and glycosylated cells), but also of the glycoproteins, eg as drugs and diagnostic agents, or Provided for use in medicine in a diagnostic kit. The method for preparing the sugar-peptide conjugate is as follows: cyclic carbamate (1) (where R ³ and R ⁴ are hydroxyl, acetamide, or carbohydrate moiety; and R ⁵ is hydrogen, methyl, hydroxymethyl, acetamide Methyl, carboxyl, or X— (CH ₂ ) _r —, where X is a carbohydrate moiety, and r is an integer selected from 0, 1, 2, and 3). Reacting with a peptide containing a secondary amino group.

Description

  The present invention not only relates to relevant glycosylcarbamoylation methods suitable for the preparation of novel glycoproteins and glycopeptides (especially glycoproteins and glycosylated cells), but also such sugars, for example as drugs and diagnostic agents or in diagnostic kits Providing the use of proteins in pharmaceuticals.

  Techniques suitable for the preparation of complex carbohydrates such as glycopeptides, glycoproteins, glycolipids, glycosylated cell surfaces, glycosylated cell membranes, and other glycosylated non-biological surfaces include drug development and glycobiology. Very important to learning. The technology that makes this possible plays an essential role in the development of glycopharmaceuticals by conjugating immunogenic and non-immunogenic carbohydrate moieties with chemical and / or biological substances. The most advanced technology is usually based on the use of ligation chemistry, in which assistance with protecting groups is used in conjugating the ligation probe to the target chemical and biological material. Absent.

  Several ligation methodologies have been described in scientific articles with a focus on substitution of N-terminal and lysine side chains of peptides and proteins to introduce the desired carbohydrate moiety. It is well known that primary amino functional groups are abundant in all biological samples such as all organs, skin, fur, silk, cell surfaces and can be easily expressed in synthetic polymers. Yes. Therefore, primary amine selective ligation methodologies have the greatest potential to offer many types of products in many industries.

  Primary amine ligation chemistry must provide adequate reactivity, chemical selectivity, and often satisfactory regioselectivity in water or other aqueous solutions while suppressing the formation of major byproducts. By-product formation in primary amine-specific ligation is due to the large number of amino groups, alcohols and phenolic hydroxyl groups, carboxyl groups, etc. present in both the ligation probe and the target multifunctional molecule / biological material. Occurs due to unwanted reactions in nucleophilic functional groups.

  Several primary amine-specific ligation methodologies have been introduced in the past. These ligation processes had significant drawbacks in the degree of substitution and selectivity made.

  The primary amine ligation techniques described above often result in a low degree of substitution or a low degree of chemoselectivity due to the use of highly reactive ligation probes such as mixed anhydrides. Also, in some cases, the use of an activator requires a complex purification process and reduces the purity of the product (mixed anhydride method, reductive amination). Further, in some ligation methodologies, concentrated by-products that are toxic or difficult to remove create serious problems in the derivatization of highly reactive biological materials (2-iminomethoxymethylthio). Ligation, acyl azimuth ligation, squarelic acid ligation). In most cases, the developed methodology uses linker systems that contain artificial and / or toxic residues that limit the scope of ligation by introducing unwanted binding moieties (aryl-isothiocyanates). Coupling with squalene ligation).

  Therefore, there is a need to develop new and appropriate ligation methodologies for conjugating carbohydrates to proteins, taking into account limited current methodologies. The new methodology must meet the following criteria:

The ligation reaction preferably takes place in an aqueous solution, preferably in water.

-Form a direct bond at the junction, preferably without an artificial linker.

-Natural non-toxic linker moieties are acceptable.
-Coupling reagents must not be used during the ligation reaction.
-The formation of concentrated by-products must be avoided.
Ligation chemistry must work over a wide pH range.
-Ligation probe reactivity must support both chemical and regioselectivity while achieving fast conjugation.

  EP-A-441192 A2 discloses retroisosteric peptides and their use as renin inhibitors.

  WO 88 / 02756A2 discloses sugar derivatives of biologically active peptides with extended duration of action.

  The present invention provides a desired ideal ligation method for coupling unprotected carbohydrates directly to peptides / proteins / biological substances in water, in a wide pH range and without using any coupling reagents. provide. In addition, the newly developed method has good chemistry and regioselectivity.

  Thus, one aspect of the present invention relates to a method for preparing a carbohydrate-peptide complex referred to in claim 1.

  Another aspect of the present invention relates to a carbohydrate-peptide complex as referred to in claims 7, 8 and 10.

  A third aspect of the present invention relates to the use of such a carbohydrate-peptide conjugate as referred to in claim 15 as a medicament.

  A fourth aspect of the present invention relates to the use of a carbohydrate-peptide complex as referred to in claim 17, as a drug, diagnostic agent or in a diagnostic kit.

  The fifth aspect of the present invention relates to a novel cyclic carbamate of an oligosaccharide.

Scheme of specific reaction of ligation of carbohydrate cyclic carbamate using disaccharide ligation probe. Scheme of specific reactions for ligation of carbohydrate cyclic carbamates using a disaccharide ligation probe for glycosylation of human insulin. Scheme of specific reaction of ligation of carbohydrate cyclic carbamate to tumor cells using a disaccharide ligation probe. General conjugation of trisaccharide to tumor cells. Preparation of lactose sugar cyclic carbamate. Preparation of carbohydrate N, O-cyclic carbamate using phosphinimine intermediate. Preparation of carbohydrate N, O-cyclic carbamate, an immunogenic carbohydrate. Preparation of carbohydrate N, O-cyclic carbamates via intramolecular cyclization of acyclic carbamates. Preparation of derivatized carbohydrate N, O-cyclic carbamate. Preparation of radiolabeled trisaccharide using carbohydrate N, O-cyclic carbamate. FIGS. 11 and 12 Staining of cell smears with FITC-labeled GS1B4. FIGS. 11 and 12 Staining of cell smears with FITC-labeled GS1B4.

（ここで、Ｒ^３及びＲ^４は、独立して、ヒドロキシル、アセトアミド、及び糖質部分からなる群から選択され；及びＲ^５は、水素、メチル、ヒドロキシメチル、アセトアミドメチル、カルボキシル、及びＸ−（ＣＨ_２）_ｒ−からなる群から選択され、ここでＸは糖質部分であり、ｒが０、１、２、及び３から選択される整数である；）
と、少なくとも１つの１級アミノ基を含むペプチドとを反応させるステップを含む、とりわけ糖質−ペプチド複合体の調製方法に関する。 As mentioned above, the present invention provides that the method comprises a cyclic carbamate (4)

(Where R ³ and R ⁴ are independently selected from the group consisting of hydroxyl, acetamide, and carbohydrate moieties; and R ⁵ is hydrogen, methyl, hydroxymethyl, acetamidomethyl, carboxyl, and X— (CH ₂ ) _r — selected from the group consisting of wherein X is a carbohydrate moiety and r is an integer selected from 0, 1, 2, and 3;)
In particular, the present invention relates to a method for preparing a carbohydrate-peptide complex comprising the step of reacting a peptide containing at least one primary amino group.

Chemical modification of biological and chemical substances is the most important reaction that can provide products characterized by new physical, chemical, biological, and physiological properties. One of the most desirable structural modifications of biopolymers and biological materials is glycosylation.
Complex carbohydrates are peptides / proteins and natural structures that provide new properties on living cell surfaces. For example, a carbohydrate-peptide complex can stabilize the peptide under investigation (eg, protein) in an optimal conformation, thereby maintaining the desired function. Carbohydrate-peptide conjugates can also increase half-life, water solubility, and promote protein stability. Covalently attached carbohydrates can also be immunodeterminants on viral surfaces, prokaryotic cells and eukaryotic cells. Some carbohydrate moieties are known to inhibit microbial adhesion, while others act as receptors for their binding. Therefore, glycoconjugates play an important role in viral and bacterial infections, and certain derivatives could be used as anti-infectives.

  Thus, in this context, the term “carbohydrate-peptide conjugate” is meant to mean a carbohydrate and peptide, eg, a complex of general formulas 1 and 2 as outlined below. Intended (see below). It should be understood that the complex includes one or more carbohydrate moieties and peptide moieties. Such carbohydrate moieties can themselves include mono-, di-, or oligosaccharides.

  Indeed, the term “carbohydrate moiety” (also referred to as a glycosyl moiety) —as used herein—but not limited to, derivative and non-derivative mono-, di-, oligo- It is intended to encompass sugars, N-, S-, and C-glycosides. The carbohydrate moiety can represent a linear or branched (often highly branched) structure of monosaccharide units. Some more commonly used monosaccharide units include glucose, N-acetyl-glucosamine, mannose, galactose, neuraminic acid, N-acetyl-neuraminic acid, and the like.

  The term “peptide” —as used herein—includes from small peptides such as oligopeptides having 5 or more amino acid units to polypeptides and proteins having 30 or more amino acid units. Intended. Typically, the peptide / peptide moiety comprises a total of at least 30 amino acid units, and typically alpha amino acids are linked by amide bonds (peptide bonds). More interestingly, for example, the total amino acid unit typically consists of at least 60, such as at least 100, or even at least 150, as a more interesting aspect. Larger peptide / peptide moieties may consist of two or more domains involved in the formation of biological peptides / proteins, such as enzymes, therapeutically related proteins, and the like.

  Cyclic carbamate (4) is a key reactant in the formation of carbohydrate-peptide complexes.

In the cyclic carbamate, R ³ and R ⁴ are independently selected from the group consisting of hydroxyl, acetamide, and carbohydrate moieties. Further, R ⁵ is selected from the group consisting of hydrogen, methyl, hydroxymethyl, acetamidomethyl, carboxyl, and X— (CH ₂ ) _r —, where X is a carbohydrate moiety and r is 0, An integer selected from 1, 2, and 3.

It should be understood that the cyclic carbamate is a di-, tri- or oligosaccharide, ie at least one of R ³ , R ⁴ , and X is a carbohydrate moiety.

（ここで、Ｒ^６及びＲ^７は、上述のＲ^３及びＲ^４において定義された通りであり、Ｒ^９は、上述のＲ^５において定義された通りであり、及びＲ^８及びＲ^１０は独立して、ヒドロキシル、Ｃ_１−６−アルコキシ、Ｃ_２−２０−アシルオキシ、アセトアミド、及び糖質部分からなる群から選択される。）
において、１、２−Ｎ、Ｏ−位の還元末端に環状カルバメート部分を有する化合物である。 Examples of interesting variants of cyclic carbamates include di- and oligosaccharides (4a), (4b), and (4c):

(Where R ⁶ and R ⁷ are as defined in R ³ and R ⁴ above, R ⁹ is as defined in R ⁵ above, and R ⁸ and R ¹⁰ are independent) And selected from the group consisting of hydroxyl, C _1-6 -alkoxy, C _2-20 -acyloxy, acetamide, and a carbohydrate moiety.)
In which the cyclic carbamate moiety is present at the reducing end of the 1,2-N, O-position.

The term “C _1-6 -alkoxy” refers to “C _1-6 -alkyl-oxy”, where “C _1-6 -alkyl” refers to 1 to 6 carbon atoms, eg methoxy, ethoxy, propyloxy, iso It is intended to mean a linear or branched hydrocarbon group which is propyloxy, butyloxy, pentyloxy, and hexyloxy.

The term “C _2-20 -acyloxy” refers to “C _1-19 -alkyl-C (═O) —O—”, where “C _1-19 -alkyl” refers to 1 to 19 carbon atoms, for example acetyl In addition to oxy, ethylcarbonyloxy, propylcarbonyloxy, iso-propylcarbonyloxy, butylcarbonyloxy, pentylcarbonyloxy, octylcarbonyloxy and the like, as well as their unsaturated species such as “C _2-20 -acyloxy” _C1-19 -alkylene-C (= O) -O- "," _C1-19 -alkyl-di-ene-C (= O) -O- "," _C1-19 -alkyl-tri-ene. -C (= O) -O- "," _C1-19 -alkyl-tetra-ene-C (= O) -O- "," _C1-19 -alkynyl-C (= O) -O- ". Etc. It refers to a chain or branched hydrocarbon group.

Reaction of Cyclic Carbamates with Peptides Peptide associates in this context have at least one primary amine group that undergoes reaction with cyclic carbamates. Certain peptides may contain several primary amines, such as side chain primary amines originating from lysine amino acid units and N-terminal primary amines originating from various amino acids (except proline). Will be understood.

  The step of reacting the cyclic carbamate (4) with the peptide involves reacting the chemical species under conditions that facilitate the reaction of one or more primary amino groups and the corresponding number of cyclic carbamate molecules.

により説明される。 The ligation reaction is the following reaction scheme (where (4) is a 1,2-N, O-cyclic carbamate (R ³ , R ⁴ and R ⁵ are as defined above); 2) is one primary amino group (H ₂ N) of the peptide (E ¹ ), and the resulting carbohydrate-peptide complex is represented by (3)):

Explained by

  In the ring-opening reaction of N-nucleophiles of primary amines of carbohydrate cyclic carbamate (4) found in peptides / proteins, biological materials, etc., (2) is a key chemical for a novel ligation process. Ligation is a powerful technique when the cyclic carbamate (4) is characterized by a trans-trans fused bicyclic system. Such extremely distorted ring systems prefer to be stabilized via a ring opening process. The preparation of a cyclic carbamate such as (4) will be described later.

Chemoselective and site-selective novel ligation reactions of corresponding carbohydrate cyclic carbamates with chemical and biological materials having primary amino groups are typically 0-40 ° C, acidic, neutral Or under basic reaction conditions, either in organic or aqueous solutions. Solvents include, but are not limited to, methanol, water, ethanol, acetone, toluene, benzene, 1,4-dioxane, DMF, pyridine, and the like, and mixtures thereof. Basic materials such as inorganic / organic bases—especially N, N-diisopropylethylamine, triethylamine, etc.—and their salts may be preferred to control pH, catalytic reaction, and regioselectivity during the substitution reaction. . Acidic substances such as inorganic / organic acids—preferably hydrochloric acid, acetic acid, formic acid etc.—and their salts such as NaH ₂ PO ₄ can be used. The reaction time for the substitution reaction is typically 3-24 hours, but depends on the structure of the substrate, the set temperature and the cyclic carbamate that is the carbohydrate. Complex carbohydrate conjugate products are typically obtained in yields ranging from 80 to 95%.

  It is important to emphasize that the regioselectivity in substitution between the N-terminus and the lysine side chain is easily maintained by setting pH adaptation and appropriate reaction conditions. Therefore, ligation reactions usually show good N-terminal selectivity at pH 4-7. Most conjugation of lysine side chains is obtained at high pH 8-10 conjugation. The new ligation is effective at pH 7 in water without any additional base / acid or any other activator. In addition, the product has glycosylurea linkages, which are believed not to be toxic or harmful to the body.

  Thus, according to one aspect of the present invention, the reaction can be performed in a polar solvent such as water.

  In one embodiment, the process of the present invention is carried out at a reaction pH in the range of 6.5-10.5. Alternatively, the reaction has a pH in the range of 4.0-7.0 to promote selective ligation at the N-terminus, or a pH of 8.0-10.0 to promote selective ligation at the lysine side chain. It is done in the range.

  This method can be applied to all types of peptides, such as single-chain peptides, multi-chain peptides, folded peptides, aggregated peptides, cell surface binding proteins, cell membrane binding proteins, and the like.

  In one particularly interesting aspect, the peptide is a cell surface or cell membrane bound protein.

  This method includes large amounts of new carbohydrate-peptide conjugates, preferably those defined and described below (see “New Carbohydrate-Peptide Complexes”).

Novel carbohydrate-peptide conjugates One class of novel carbohydrate-peptide conjugates is defined in general formula 1 where one or more carbohydrate moieties are present at the glycoside site at the lysine residue of the peptide / protein. The ε-amino function of the group is linked via a carbonyl linker.

（ここで、
Ｒ^１及びＲ^２は、間に存在するリジン部分と一緒になって、ペプチド部分を表し；
Ｒ^３及びＲ^４は、独立して、ヒドロキシル、アセトアミド、及び糖質部分からなる群から選択され；
Ｒ^５は、水素、メチル、ヒドロキシメチル、アセトアミドメチル、カルボキシル、及びＸ−（ＣＨ_２）_ｒ−からなる群から選択され、ここで、Ｘは糖質部分であり、ｒは０、１、２、及び３から選択される整数である；）
の１つ以上の部分を含む糖質−ペプチド複合体、及び、それらの薬学的に許容される塩に関する。 Thus, the present invention also provides a general formula 1:

(here,
R ¹ and R ² together with the lysine moiety present in between represent a peptide moiety;
R ³ and R ⁴ are independently selected from the group consisting of hydroxyl, acetamide, and carbohydrate moieties;
R ⁵ is selected from the group consisting of hydrogen, methyl, hydroxymethyl, acetamidomethyl, carboxyl, and X— (CH ₂ ) _r —, where X is a carbohydrate moiety and r is 0, 1, 2, , And an integer selected from 3;)
To carbohydrate-peptide conjugates comprising one or more moieties thereof, and pharmaceutically acceptable salts thereof.

（ここで、Ｒ^６及びＲ^７は、上述のＲ^３及びＲ^４において定義された通りであり、及びＲ^９は上述のＲ^５において定義された通りであり、及びＲ^８は、ヒドロキシル、Ｃ_１−６−アルコキシ、Ｃ_２−２０−アシルオキシ、アセトアミド、及び糖質部分からなる群から選択される。）
のいずれか１つ以上の部分を含む糖質−ペプチド複合体である。 Examples shown here are general formulas 1a, 1b, and 1c.

(Where R ⁶ and R ⁷ are as defined above for R ³ and R ⁴ , and R ⁹ is as defined above for R ⁵ , and R ⁸ is hydroxyl, C Selected from the group consisting of _1-6 -alkoxy, C _2-20 -acyloxy, acetamide, and a carbohydrate moiety.)
It is a carbohydrate-peptide complex containing any one or more of these.

  A different class of novel carbohydrate-peptide conjugates is defined by general formula 2, where the carbohydrate moiety is linked via a carbonyl linker at the N-terminal amino function of the peptide / protein and at the glycoside site. .

（ここで、
Ｒ^１１はアミノ酸側鎖；
Ｒ^２は−ＮＨ−ＣＨＲ^１１−Ｃ（＝Ｏ）−と一緒になって、合計のアミノ酸が少なくとも３０のペプチド部分を表し；
Ｒ^３及びＲ^４は、独立して、ヒドロキシル、アセトアミド、及び糖質部分からなる群から選択され；
Ｒ^５は、水素、メチル、ヒドロキシメチル、アセトアミドメチル、カルボキシル、及びＸ−（ＣＨ_２）_ｒ−からなる群から選択され、ここでＸは糖質部分であり、ｒは０、１、２及び３から選択される整数である；）
の１つ以上の部分を含む糖質−ペプチド複合体、及び、それらの薬学的に許容される塩に関する。 Thus, the present invention also provides a general formula 2:

(here,
R ¹¹ is an amino acid side chain;
R ² together with —NH—CHR ¹¹ —C (═O) — represents a peptide moiety with a total of at least 30 amino acids;
R ³ and R ⁴ are independently selected from the group consisting of hydroxyl, acetamide, and carbohydrate moieties;
R ⁵ is selected from the group consisting of hydrogen, methyl, hydroxymethyl, acetamidomethyl, carboxyl, and X— (CH ₂ ) _r —, where X is a carbohydrate moiety and r is 0, 1, 2, and An integer selected from 3;)
To carbohydrate-peptide conjugates comprising one or more moieties thereof, and pharmaceutically acceptable salts thereof.

（ここで、Ｒ^６及びＲ^７は、上述のＲ^３及びＲ^４において定義された通りであり、Ｒ^９は上述のＲ^５において定義された通りであり、及びＲ^８は、ヒドロキシル、Ｃ_１−６−アルコキシ、Ｃ_２−２０−アシルオキシ、アセトアミド、及び糖質部分からなる群から選択される。）
のいずれか１つ以上の部分を含む糖質−ペプチド複合体である。 Examples shown here are general formulas 2a, 2b, and 2c.

(Where R ⁶ and R ⁷ are as defined above for R ³ and R ⁴ , R ⁹ is as defined above for R ⁵ , and R ⁸ is hydroxyl, C _{1 -6} -alkoxy, C _2-20 -acyloxy, acetamide, and selected from the group consisting of carbohydrate moieties.)
It is a carbohydrate-peptide complex containing any one or more of these.

  The classes of carbohydrate-peptide complexes represented by the above general formula 1 and general formula 2 are partially overlapping, and one or more carbohydrate moieties are ε- of the lysine residue of the peptide at the glycoside site. It is linked to the amino functional group via a carbonyl linker, and-in the same peptide-the carbohydrate moiety is easily linked to the N-terminal amino functional group at the glycoside site via the carbonyl linker. It will be understood that it is expected. If the peptide consists of -2 chains or more, it may contain -2 or more N-terminally linked carbohydrate moieties.

  In some interesting embodiments, the peptide is a cell surface or cell membrane bound protein.

  The term “amino acid side chain” typically refers to a side chain group of amino acids in a peptide (including synthetic peptides) and is not intended to be limited to 20 essential amino acids. Examples of amino acid side chains are hydrogen (representing glycine), methyl (alanine), 2-propyl (valine), 2-methyl-1-propyl (leucine), 2-butyl (isoleucine), methylthioethyl (methionine), Benzyl (phenylalanine), 3-indolylmethyl (tryptophan), hydroxymethyl (serine), 1-hydroxyethyl (threonine), mercaptomethyl (cysteine), 4-hydroxybenzyl (tyrosine), aminocarbonylmethyl (asparagine), 2 -Aminocarbonylethyl (glutamine), carboxymethyl (aspartic acid), 2-carboxyethyl (glutamic acid), 4-amino-1-butyl (lysine), 3-glutathion-1-propyl (arginine), and 4-imidazolylmethyl (Histidine) That.

The term “pharmaceutically acceptable salts” is intended to include acid addition salts and basic salts. Illustratively, acid addition salts are pharmaceutically acceptable salts formed with non-toxic acids. Examples of such organic salts are their maleic acid, fumaric acid, benzoic acid, ascorbic acid, succinic acid, oxalic acid, bis-methylenesalicylic acid, methanesulfonic acid, ethanedisulfonic acid, acetic acid, propionic acid, tartaric acid, salicylic acid , Citric acid, gluconic acid, lactic acid, malic acid, mandelic acid, cinnamic acid, citraconic acid, aspartic acid, stearic acid, palmitic acid, itaconic acid, glycolic acid, p-aminobenzoic acid, glutamic acid, benzenesulfonic acid, and theophylline There are not only acetic acid, but also salts of 8-halotheophylline acetic acid, such as 8-bromotheophylline acetic acid. Examples of such inorganic acid salts are their hydrochloric acid, hydrogen bromide, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid salts. A basic salt is one in which the counter ions (residual) are alkali metals such as sodium and potassium, alkaline earth metals such as calcium and ammonium ions ( ⁺ N (R) ₃ R ′, where R and R Is independently selected from optionally substituted C _1-6 -alkyl, optionally substituted C _2-20 -alkenyl, optionally substituted aryl, or optionally substituted heteroaryl. Salt. Pharmaceutically acceptable salts are described, for example, by Remington's Pharmaceutical Sciences, 17. Ed. Alfonso R. Gennaro (Ed.), Mack Publishing Company, Easton, PA, U.S., 198. The latest edition and those described in Encyclopedia of Pharmaceutical Technology. Therefore, the term “acid addition salts and basic salts thereof” is intended to include such salts herein. Furthermore, not only compounds but any intermediate or starting material may exist in the form of hydrates.

  Furthermore, it will be appreciated that the compounds may exist in the form of a racemic mixture or individual stereoisomers, such as enantiomers or diastereomers. The present invention encompasses not only possible stereoisomers (eg enantiomers and diastereomers), but also racemic and mixtures that increase one of the possible stereoisomers.

Preparation of cyclic carbamates Preparation of cyclic carbamate derivatives (4) (and (4a), (4b) and (4c)) can be accomplished by glycosyl azide or other corresponding azido-deoxy-oligosaccharide derivatives in the presence of trialkyl / arylphosphine. Under the treatment with carbon dioxide. Typically, the reaction is carried out in an anhydrous organic solution in the range of 0-40 ° C. under neutral reaction conditions. Solvents include, but are not limited to, acetone, toluene, benzene, 1,4-dioxane, DMF, tetrahydrofuran and the like and mixtures thereof. The reaction time for cyclic carbamate formation is typically 3-24 hours depending on the structure of the substrate, the set temperature, and the nature of the trialkyl / aryl phosphine used. Cyclic carbamate products are typically obtained in high yields of 80-95%.

  Alternatively, the preparation of the cyclic carbamate derivatives (4) (and (4a), (4b) and (4c)) can be carried out with glycosyl azides or other corresponding azido-deoxy-oligosaccharide derivatives, including isolation of phosphinimine derivatives. , Based on treatment with trialkyl / arylphosphine. In the second step, the phosphinimine derivative is reacted with carbon dioxide to give the desired oligosaccharide cyclic carbamate. Typically, the reaction is carried out in the range of 0-40 ° C under neutral conditions. Solvents include, but are not limited to, acetone, toluene, benzene, 1,4-dioxane, DMF, tetrahydrofuran and the like and mixtures thereof. The reaction time for phosphine formation is 3-10 hours depending on the nature of the trialkyl / aryl phosphine used. Cyclic carbamate formation is typically 3-24 hours depending on the structure of the substrate and the set temperature. Cyclic carbamate products are typically obtained in high yields of 80-95%.

  Alternatively, the preparation of the cyclic carbamate derivatives (4) (and (4a), (4b) and (4c)) can be accomplished using glycosylamines or other amino-deoxy-oligosaccharides as phosgene or other suitable phosgene derivatives such as diphosgene. Or with treatment with triphosgene. Typically, the reaction is carried out in an organic or water solution in the range of 0-40 ° C. under neutral or basic reaction conditions. Solvents include, but are not limited to, acetone, toluene, benzene, 1,4-dioxane, DMF, tetrahydrofuran, pyridine, and the like, and mixtures thereof. The reaction time for cyclic carbamate formation is typically 3-24 hours depending on the structure of the substrate and the nature of the set temperature. Cyclic carbamate products are typically obtained in high yields of 80-95%.

  Alternatively, preparation of cyclic carbamate derivatives (4) (and (4a), (4b) and (4c)) can be achieved by treating the acyclic carbamate derivative with a base such as sodium hydride or DBU, Is based on starting. Typically, the reaction is carried out in anhydrous organic solution under basic conditions in the range of 0-40 ° C. Solvents include, but are not limited to, acetone, toluene, benzene, 1,4-dioxane, DMF, tetrahydrofuran and the like and mixtures thereof. The reaction time for cyclic carbamate formation is 3-24 hours, depending on the structure of the substrate and the set temperature. Cyclic carbamate products are typically obtained in high yields of 80-95%.

  Cyclic carbamate derivatives (4) (and (4a), (4b) and (4c)) can undergo, for example, O-acylation, O-alkylation, cyclic acetal, cyclic ketal formation, unprotected under multiple reaction conditions. It is important to emphasize that it has good stability in wide chemical induction such as hydrolysis of derivatives. Such unique options may broaden the use of cyclic carbamate ligation probes and allow multiple inducers to be ligated to peptides / proteins, biological and complex chemicals.

（ここで、Ｒ^６及びＲ^７は、上述のＲ^３及びＲ^４において定義された通りであり、及びＲ^９は上述のＲ^５において定義された通りである。Ｒ^８及びＲ^１０は、独立して、ヒドロキシル、Ｃ_１−６−アルコキシ、Ｃ_１−６−アシルオキシ、アセトアミド、及び糖質部分からなる群から選択される。）
は、そのような新規なものであり、それゆえ本発明はそのような物質に関わり、ここで、これらは、とりわけ一般式２ａ、２ｂ、２ｃ、３ａ、３ｂ、及び３ｃの化合物の調製の中間体として有用であると信じられている。 Oligosaccharide cyclic carbamates (4a), (4b) and (4c), ie

(Where R ⁶ and R ⁷ are as defined above for R ³ and R ⁴ , and R ⁹ is as defined above for R ^5. R ⁸ and R ¹⁰ are independent And selected from the group consisting of hydroxyl, C _1-6 -alkoxy, C _1-6 -acyloxy, acetamide, and a carbohydrate moiety.)
Are such novel and therefore the present invention relates to such materials, where these are inter alia intermediate in the preparation of compounds of general formulas 2a, 2b, 2c, 3a, 3b and 3c. It is believed to be useful as a body.

Uses of carbohydrate-peptide conjugates In general, the uses of carbohydrate-peptide conjugates as defined herein are as drugs, diagnostic agents, or in diagnostic kits.

  In particular, the carbohydrate-peptide conjugates defined and described herein, including those prepared according to the methods defined and described herein, are believed to provide many possibilities in medicine. It has been.

  As one variant, the glycosyl moiety (moiety / moieties) (carbohydrate moiety) of a carbohydrate-peptide complex represents a non-immunogenic carbohydrate.

  In one preferred embodiment, some non-immunogenic carbohydrates such as dextran, maltodextrin, maltose, cellobiose, an O-methylated oligosaccharide, thio-linked oligosaccharides increase the half-life. And can be coupled to therapeutic peptides / proteins to provide improvements in beneficial physics, biology, and physiological properties such as solubility, heat and enzyme stability.

  In other variants, the glycosyl moiety (carbohydrate moiety) of the carbohydrate-peptide complex represents an immunogenic carbohydrate.

In the variant, several immunogenic carbohydrates such as ABO blood antigen, Lewis antigen, tumor specific antigen, α-Gal-epitope, α-mannosyl-epitope, polysialic acid may bind to:
-Peptides / proteins for down-regulating peptides / proteins with activated vaccines.
-Viruses such as HIV, hepatitis B, herpes, influenza, avian influenza, etc. (live vaccines, inactivated vaccines, or virus particles) to produce vaccines to combat infection.
-Bacteria for the preparation of mycobacteria, Helicobacter etc. for antibacterial vaccines.
-Tumor cells to modify immunological properties and generate a strong immune response with autologous cancer vaccines.
-Tumor cell membranes for preparing autologous cancer vaccines.

As a further aspect, fat-soluble oligosaccharides can be used to improve peptide / protein stability by adhering to albumin.
Part 1: Ligation

Maltosyl cyclic carbamate 1 (40 mg) and BSA (bovine serum albumin, 50 mg) 2 were dissolved in water (5 mL) (see FIG. 1). Triethylamine and acetic acid were added to adjust the pH to about 9.44. The mixture was maintained at room temperature for 4 hours, after which the reaction mixture was transferred to a dialysis membrane and dialyzed against distilled water for 2 days, after which lyophilized product 3 was obtained as a white powder.
Mass spectrometry: MALDI-TOF: glycosylated BSA 68333, BSA standard: 66134.

Maltosyl cyclic carbamate 1 (40 mg) and BSA (bovine serum albumin, 50 mg) 2 were dissolved in water (5 mL) (see FIG. 1). Triethylamine and acetic acid were added to adjust the pH to about 8.57. The mixture was maintained at room temperature for 4 hours, after which the reaction mixture was transferred to a dialysis membrane and dialyzed against distilled water for 2 days, after which lyophilized product 3 was obtained as a white powder.
Mass spectrometry: MALDI-TOF: glycosylated BSA69099, BSA standard: 66134.

Maltosyl cyclic carbamate 1 (40 mg) and BSA (bovine serum albumin, 50 mg) 2 were dissolved in water (5 mL) (see FIG. 1). Triethylamine and acetic acid were added to adjust the pH to about 8.10. The mixture was maintained at room temperature for 4 hours. The reaction mixture was then transferred to a dialysis membrane and dialyzed against distilled water for 2 days, after which lyophilized product 3 was obtained as a white powder.
Mass spectrometry: MALDI-TOF: glycosylated BSA 68329, BSA standard: 66134.

Maltosyl cyclic carbamate 1 (40 mg) and BSA (bovine serum albumin, 50 mg) 2 were dissolved in water (5 mL) (see FIG. 1). Triethylamine and acetic acid were added to adjust the pH to about 7.45. The mixture was maintained at room temperature for 4 hours. The reaction mixture was then transferred to a dialysis membrane and dialyzed against distilled water for 2 days, after which lyophilized product 3 was obtained as a white powder.
Mass spectrometry: MALDI-TOF: glycosylated BSA67679, BSA standard: 66134.

Maltosyl cyclic carbamate 1 (10 mg) and human insulin 4 (50 mg) were dissolved in water (13 mL) (see FIG. 2). Diisopropylethylamine and aqueous NaH ₂ PO ₄ were added to adjust the pH to about 10.00. The mixture was maintained at room temperature for 2.5 hours and the reaction mixture was lyophilized to give B29 glycosylated insulin 5 with regioselectivity greater than 90%.
Mass spectrometry: MALDI-TOF: B-29-glycosylated human insulin: 6171, human insulin: 5805.

Maltosyl cyclic carbamate 1 (10 mg) and human insulin 4 (50 mg) were dissolved in water (13 mL) (see FIG. 2). Diisopropylethylamine and aqueous NaH ₂ PO ₄ were added to adjust the pH to about 8.00. The mixture was maintained at room temperature for 2.5 hours and the reaction mixture was lyophilized to give modified insulin 5.
Mass spectrometry: MALDI-TOF: glycosylated human insulin: 6171, human insulin: 5804.

Maltosyl cyclic carbamate 1 (10 mg) and human insulin 4 (50 mg) were dissolved in water (13 mL) (see FIG. 2). Diisopropylethylamine and aqueous NaH ₂ PO ₄ were added to adjust the pH to about 7.00. The mixture was maintained at room temperature for 2.5 hours and the reaction mixture was lyophilized to give B-1-glycosylated insulin 5 with regioselectivity greater than 90%.
Mass spectrometry: MALDI-TOF: B-1-glycosylated human insulin: 6171, human insulin: 5804.

α-Gal epitope-cyclic carbamate 6 (disaccharide) (25 mg) and human breast cancer cell line 7 (2 × 10 ⁶ cells) were mixed in PBS buffer (5 mL) (see FIG. 3). The reaction mixture was maintained at 37 ° C. for 3 hours, after which the mixture was centrifuged to separate the cells from the medium.
Modified cells were treated with fluorescent label-lectin and the modification was verified by flow cytometry.

α-Gal epitope-carbamate 6 (disaccharide) (25 mg) and human breast cancer cell line 7 (cell number 1 × 10 ⁶ ) were mixed in PBS buffer (5 mL) (see FIG. 3). The reaction mixture was maintained at 37 ° C. for 3 hours, after which the mixture was centrifuged to separate the cells from the medium.
Modified cells were treated with fluorescent label-lectin and the modification was verified by flow cytometry.

α-Gal epitope-carbamate 6 (disaccharide) (25 mg) and human breast cancer cell line 7 (5 × 10 ⁵ cells) were mixed in PBS buffer (5 mL) (see FIG. 3). The reaction mixture was maintained at 37 ° C. for 3 hours, after which the mixture was centrifuged to separate the cells from the medium.
Modified cells were treated with fluorescent label-lectin and the modification was verified by flow cytometry.
In Examples 11 to 15, Compound 9 is referred to as the “trisaccharide probe”. See FIG. In Examples 14 and 15, the marked carbon atom was labeled with C13.

Conjugation of trisaccharide probe to B16BL16 cell line
Cell preparation B16. Confluent in DMEM containing 10% fetal calf serum (no P / S) at 37.5 ° C., 5% CO ₂ . Two flasks (300 cm ² ) of BL6 melanoma cells (TPP, Trasadingen Switzerland) were used in this experiment. Growth medium was removed from the cells and 25 mL PBS (without Mg ²⁺ and Ca ²⁺ ) (Gibco Invitrogen, Taastrup Denmark) was added to the cells and immediately removed again. Another 10 mL of PBS was added and removed, and the cells were harvested with cell detachment solution C5914 (Sigma-aldrich, Saint Louis Missouri) (3 mL / flask) and transferred to a 10 mL tube (TPP, Trasingingen Switzerland). The cells were centrifuged at 300 G for 5 minutes at 20 ° C. and the supernatant was discarded. Subsequently, the cells were washed with PBS (without Mg ²⁺ and Ca ²⁺ ), centrifuged as described above, and resuspended in PBS (without Mg ²⁺ and Ca ²⁺ ). The concentration of the cell suspension was determined.

B16 of the α-gal epitope. Conjugation to BL6 melanoma cells 500 mg α-Gal trisaccharide-carbamate (9) was dissolved in PBS (without Mg ²⁺ and Ca ²⁺ ) (Gibco Invitrogen, Taastrup Denmark) and filtered through a sterile filter (Sartorius Miniisart®) ), 0.20 my) (Sartorius, Goettingen Germany). Eight different concentrations of the complex were prepared. 2mL of PBS ^(Mg None ²⁺ and ^{Ca 2+)} and 10 ⁶ of B16. To a flask containing 600 μL of cell suspension containing BL6 cells, 2 mL of each complex dilution was added. The final volume of the flask was 4.6 mL. The flask was numbered 1-10. Flasks 1 and 10 contained no complex and were used as controls. The cells were cultured with the complex at 37 ° C., 5% CO ₂ for 3 hours and 25 minutes.

After conjugation, cells (non-conjugated cells) from the conjugation reactions 1, 2, 4, 6, and 8 were harvested with 10 mL of cell detachment solution C5914 (Sigma-Aldrich, Saint Louis Missouri) and 10 mL tubes (TPP, (Tradadingen Switzerland). Cells were centrifuged at 300 G for 5 minutes at 20 ° C. and resuspended in PBS (without Mg ²⁺ and Ca ²⁺ ). Finally, the cells were centrifuged as described above and resuspended in 100 μL TBS containing 1% BSA. Cell concentration and viability were determined by microscopy using trypan blue stained cell samples. Cells from conjugation reactions 3, 5, 7, 9, and 10 (non-conjugated cells) were harvested as described above the next day. The cells were centrifuged at 300 G for 5 minutes at 20 ° C. and resuspended in 100 μL of 1% BSA-containing TBS. Cell concentration and viability were determined by microscopy using trypan blue stained cell samples.

Staining of cell smears with FITC-labeled GS1B4 Cell smears from conjugate reaction 1 (non-conjugated cells) 2, 4, 6 and 8 were diluted for 1 hour at room temperature with a TBS1: 200 dilution of FITC-labeled GS1B4 And incubated at room temperature for 1 hour. After washing with TBS, 1 drop of Fluorescent Mounting Medium (DAKO) was added to each cell specimen, and a cover glass was placed on top. The binding of FITC-labeled GS1B4 to Galα1, 3Gal was evaluated by a fluorescence microscope.

Flow cytometry analysis B16. From conjugation reaction 1 (non-conjugated cells), 2, 4, 6 and 8. BL6 cells were analyzed by flow cytometry. 100 μL of cell suspension containing 1 × 10 ⁵ cells was cultured with 10 μg / mL (1: 100 dilution) FITC-labeled GS1B4 containing 1% BSA in TBS for 15 minutes at 4 ° C. The percentage of cell staining, mean and median cell fluorescence intensity were analyzed by flow cytometry. Thereafter, the cells were washed once with PBS and the measurement was repeated. Rabbit erythrocytes (RRBC), known to express ˜2 × 10 ⁶ Galα1,3Gal epitope / cell, were used for comparison. B16. BL6 cell conjugation and RRBC analysis were performed on different days.

Cell concentration and viability after conjugation At the end of the conjugation process, cell number and viability were assessed by trypan blue staining, which was significantly reduced in all conjugation reactions. However, there was considerable diversity between different conjugation reactions. From each conjugation reaction, a cell suspension volume of 100 μL to 130 μL was collected.

＊）細胞濃度及び生存率は、接合プロセスを行った１日後に測定した。 Table 1 Cell concentration and viability after conjugation

*) Cell concentration and viability were measured one day after the conjugation process.

Table 2 Cell viability after conjugation

Staining of cell smears with FITC-labeled GS1B4 Galα1,3Gal epitopes detected by binding to FITC-labeled GS1B4 were found in cells of all conjugation reactions tested (conjugation reactions 2, 4, 6 and 8). None of the non-conjugated cells (conjugation reaction 1) was stained.
See Figures 11 and 12.

Galα1,3Gal epitopes detected by binding to flow cytometry FITC-labeled GS1B4 were found in cells of all conjugation reactions tested (conjugation reactions 2, 4, 6, and 8). None of the non-conjugated cells (conjugation reaction 1) was stained. The percentage of cells with detectable amounts of Galα1,3Gal-epitope was determined in each conjugation reaction.

Table 3 Detectable Galα1, 3Gal-epitope cells

The fluorescence intensity of the cells having a detectable amount of cell surface Galα1, 3Gal-epitope was measured. The results showed that the number of Galα1,3Gal-epitope in conjugated cells was significantly higher than the ˜2 × 10 ⁶ Galα1,3Gal epitope known to be expressed in rabbit red blood cells. The average and median values of the cell fluorescence intensity of the washed and non-washed cells are shown below.

＊）ウサギ赤血球は、〜２×１０^６ Ｇａｌα１、３Ｇａｌエピトープ／細胞を発現することが知られている。 Table 4 Fluorescence intensity, unwashed cells

*) Rabbit erythrocytes are known to express ˜2 × 10 ⁶ Galα1, 3Gal epitope / cell.

  B16. From conjugation reaction 2. Visualization of Galα1, 3Gal epitope conjugated to BL6 melanoma cells. Cell membranes of Galα1, 3Gal epitopes on the cell surface bound with FITC-labeled GS1B4 were firmly stained.

Table 5 Fluorescence intensity, washed cells

Table 6 Fluorescence intensity, unwashed cells

B16 of α-Gal trisaccharide-carbamate probe. Conjugation to BL6 cell line.
Reduction of bonding time:
Cell Preparation See Example 11.

B16 of the α-gal epitope. Conjugated α-Gal trisaccharide-carbamate (9) to BL6 melanoma cells is dissolved in both PBS (without Mg ²⁺ and Ca ²⁺ ) (Gibco Invitrogen, Taastrup Denmark) and HBSS (with D-glucose) and with a sterile filter Filtered (Sartorius Minisart ™, 0.20 my) (Sartorius, Goettingen Germany). Three different concentrations of trisaccharide probes were prepared in both PBS and HBSS solutions.

10 ⁶ B16. Eight small flasks (25 cm ² ) containing BL6 cells (TPP, Trasadingen Switzerland) were prepared and a trisaccharide probe was added. The total volume of the flask was 4.6 mL. Cells were incubated with the complex for 1 hour at 37 ° C., 5% CO ₂ .

Table 7 Bonding reaction

  After conjugation, the supernatant from each flask was transferred to a separate 10 mL tube (TPP, Trasadingen Switzerland). Cells (non-conjugated cells) were harvested with cell detachment solution C5914 (2 mL / flask) (Sigma-Aldrich, Saint Louis, Missouri) and transferred to a 10 mL tube. Cells were centrifuged at 300 G for 5 minutes at 20 ° C. and resuspended in 0.5 mL PBS or HBSS. Cell concentration and viability were determined by microscopy using trypan blue stained cell samples.

  After 1 hour incubation with the trisaccharide probe, most cells were alive and adhered to the bottom surface of the flask. However, harvesting these cells was difficult and most were lost during the procedure.

Table 8 Cell viability

Table 9 Cell viability

B16 of α-Gal trisaccharide-carbamate probe (9). Conjugation to BL6 cell line Decrease in conjugation time:
Cell Preparation See Example 11.

B16 of the α-Gal epitope. Conjugated α-Gal trisaccharide probe (9) to BL6 melanoma cells was dissolved in HBSS (Gibco Invitrogen, Taastrup Denmark) and filtered through a sterile filter (0.2 μm Super® Acrodisc® 13, (Gelman Sciences, Ann Arbor Michigan). Three different concentrations of α-Gal trisaccharide-carbamate (9) were made. 4mL bonding solution of a total ¹⁰⁶ of B16. Five small flasks added to 606 μL of cell suspension containing BL6 cells were prepared. Each flask had a total volume of 4.6 mL. Cells were cultured with “Glycom conjugate” at 37 ° C. for 3 or 1.5 hours.

Table 10 Joining reaction

  After conjugation, the supernatant from each flask was transferred to a separate 10 mL tube (TPP, Trasadingen Switzerland). Cells were washed twice by adding PBS to the flask and immediately removed. Cells were harvested with cell detachment solution (2 mL / flask) (Sigma-Aldrich, Saint Louis Missouri). After 5 minutes, the cells were transferred to a 10 mL test tube. The cells were centrifuged at 300 G for 5 minutes at 20 ° C. and resuspended in 0.5 mL PBS. Cell concentration and viability were determined by microscopy using trypan blue stained cell samples.

Cells from flow cytometry analysis conjugation reaction 1:
100 μL of each cell suspension containing 1 × 10 ⁵ cells was cultured in a PBS solution of 10 μg / mL (1: 100 dilution) FITC-labeled GS1B4 at 4 ° C. for 15 minutes. Rabbit red blood cells (A) were used for comparison.

Cells from conjugation reactions 2, 3, 4, and 5:
The exact cell concentration was not determined because few cells were available for flow cytometric analysis. Instead, the number of cells was estimated to be 0.4 × 10 ⁵ . The cells were cultured for 15 minutes at 4 ° C. in 4 μg / mL FITC-labeled GS1B4 in PBS. Rabbit red blood cells (B) were used for comparison.
Mean and median cell fluorescence intensity was determined by flow cytometry.

Flow cytometry analysis Galα1, 3Gal epitopes detected by binding to FITC-labeled GS1B4 were found in cells of all conjugation reactions tested. Analysis of cells from conjugation reactions 2 and 4 showed that there are two populations of cells with different fluorescence intensities. The relatively high background staining indicated by the fluorescence intensity of non-conjugated cells (conjugation reaction 5) is believed to be the result of the use of a large excess of FITC-labeled GS1B4 due to overestimation of the number of cells used.

＊）ウサギ赤血球は、〜２×１０^６ Ｇａｌα１、３Ｇａｌエピトープ／細胞を発現することが知られている。 Table 11 Fluorescence intensity, cells from conjugation reaction 1

*) Rabbit erythrocytes are known to express ˜2 × 10 ⁶ Galα1, 3Gal epitope / cell.

＊）ウサギ赤血球は、〜２×１０^６ Ｇａｌα１、３Ｇａｌエピトープ／細胞を発現することが知られている。
＊＊）接合反応２及び４においては、蛍光強度の異なる２つの細胞群が見られたため、両群について値を出した。 Table 12 Cells from fluorescence intensity, conjugation reactions 2, 3, 4, and 5

*) Rabbit erythrocytes are known to express ˜2 × 10 ⁶ Galα1, 3Gal epitope / cell.
**) In the conjugation reactions 2 and 4, since two cell groups having different fluorescence intensities were observed, values were given for both groups.

B16 of radioactive probe. Conjugation to BL6 cell line The purpose of this study was to investigate the B16. The aim is to estimate the number of junctions to BL6 melanoma cells and to compare the results obtained by scintillation and flow cytometry.

Cell preparation B16. Confluent in DMEM containing 10% fetal calf serum (no P / S) at 37.5 ° C., 5% CO ₂ . Nine flasks (300 cm ² ) of BL6 melanoma cells (TPP, Trasadingen Switzerland) were used in this experiment. Growth medium was removed from the cells and 20 mL of D-glucose-containing HBSS (without Mg ²⁺ and Ca ²⁺ ) (Gibco Invitrogen, Taastrup Denmark) was added to the cells and immediately removed again. Another 10 mL of D-glucose-containing HBSS (Mg ²⁺ and no Ca ²⁺ ) was added and removed, and the cells were harvested with cell detachment solution C5914 (Sigma-aldrich, Saint Louis Missouri) (10 mL / flask) and 50 mL tube (TPP, Trasadingen Switzerland). The cells were centrifuged at 300 G for 5 minutes at 20 ° C. and the supernatant was discarded. Subsequently, the cells were washed with D-glucose-containing HBSS (without Mg ²⁺ and Ca ²⁺ ), centrifuged as described above, and resuspended in D-glucose-containing HBSS (without Mg ²⁺ and Ca ²⁺ ). The concentration of the cell suspension was determined.

Galα1, B16 of the 3Gal epitope. Conjugated radiolabels and normal trisaccharide probes to BL6 melanoma cells were dissolved in D-glucose-containing (Mg2 ⁺ and Ca2 ⁺ -free) HBSS (Gibco Invitrogen, Taastrup Denmark) and filtered through a sterile filter (0. 2 [mu] m Super (TM) Acrodisc (TM) 13, Gelman Sciences, Ann Arbor Michigan). Four different concentrations of “Glycom conjugate” were made (25 mg, 5 mg, 1 mg, and 0.1 mg). A total of 10 × 10 ⁶ B16. Twelve flasks (150 cm ² ) containing BL6 cells (TPP, Trasadingen Switzerland) were prepared. D-glucose-containing HBSS (without Mg ²⁺ and Ca ²⁺ ) was added to the trisaccharide probe, bringing the total volume of each flask to 20 mL. The cells were incubated with “Glycom conjugate” at 37 ° C. for 1.5 hours. After conjugation, the supernatant from each flask was transferred to a different 50 mL tube (TPP, Trasadingen Switzerland). Cells were harvested with cell detachment solution C5914 (5 mL / flask) (Sigma-aldrich, Saint Louis Missouri), and the supernatant was transferred to a 50 mL tube (TPP, Trasadingen Switzerland). Cells were centrifuged at 300 G for 3 minutes at 20 ° C. and resuspended in 5 mL PBS (without Mg ²⁺ and Ca ²⁺ ). This was done three times before determining cell concentration and viability by microscopic examination of trypan blue stained samples.

Cells bound to scintillation radiolabeled trisaccharide probe were centrifuged at 1000 G for 5 minutes at 20 ° C. The supernatant was discarded and the remaining pellet was dried. Subsequently, the pellet was dissolved in 400 μL of milli-Q water and 1.5 mL of scintillation solution was added. For the three control cells, standards for scintillation were added at 500 pCi, 0.5 pCi, and 0 pCi. Samples were stored at room temperature for 3 days until the cell suspension was transferred to scintillation vials and counted.

Flow cytometry analysis Cells conjugated with normal trisaccharide probes were analyzed by flow cytometry. 400 μL of each cell suspension containing 2 × 10 ⁵ cells was cultured with 10 μg / mL FITC-labeled GS1B4 at 4 ° C. for 15 minutes. The average fluorescence intensity of the cells stained with GS1B4 was measured by flow cytometry.

In the cell concentration and post-conjugation viability harvesting process, two different fractions of cells with the radiolabeled trisaccharide probe were mistakenly combined and had to be discarded. Therefore, the results obtained from these joining reactions could not be compared with others. In addition, the suspension containing cells conjugated with 0.1 mg radiolabeled trisaccharide probe resulted in an abnormal volume. As seen in the preliminary studies, the amount of harvested cells and cell viability differ between different complex concentrations and between normal and radiolabeled complexes.

＊）α−ｇａｌ接合後の評価 Table 13 Cells conjugated to normal complexes

*) Evaluation after α-gal bonding

＊）α−ｇａｌ接合後の評価
＊＊）付着した細胞のみ
＊＊＊）上清からの細胞のみ Table 14 Cells conjugated with radiolabeled complex

*) Evaluation after α-gal conjugation **) Only attached cells **) Only cells from supernatant

As seen in the flow cytometry preliminary test, there was only one fraction of FITC-labeled GS1B4 detectable amount of conjugated cells. The mean fluorescence intensity of the cells at 1 mg conjugate was unexpectedly low, whereas in the remaining conjugate reactions there was a positive correlation between the complex dose and the mean fluorescence intensity.

＊）ＧＳ１Ｂ４で標識した細胞の平均蛍光強度 Table 15 Average fluorescence intensity

*) Average fluorescence intensity of cells labeled with GS1B4

A scintillation positive correlation was seen in scintillation counts and conjugate dose. However, due to the above mentioned errors in the harvesting process, the results obtained from flow cytometry analysis could not be compared and therefore the relationship between the two quantification methods could not be determined.

＊）付着した細胞のみ
＊＊）上清からの細胞のみ Table 16 Epitope / cell estimates

*) Only attached cells **) Only cells from the supernatant

Table 17 Epitope / cell as a function of conjugation amount

Radioactive probe and B16. Conjugation of the BL6 cell line The purpose of this study was to investigate the B16. The aim is to estimate the number of junctions to BL6 melanoma cells and to compare the results obtained by scintillation and flow cytometry.

Preparation of cells B16. Confluent in DMEM containing 10% fetal calf serum (no P / S) at 37.5 ° C., 5% CO ₂ . Twelve flasks (300 cm ² ) of BL6 melanoma cells (TPP, Trasadingen Switzerland) were used in this experiment.
Growth medium was removed from the cells and 20 mL of D-glucose-containing HBSS (without Mg ²⁺ and Ca ²⁺ ) (Gibco Invitrogen, Taastrup Denmark) was added to the cells and immediately removed again. Another 10 mL D-glucose containing (Mg ²⁺ and no Ca ²⁺ ) HBSS is added and removed, and the cells are harvested with cell detachment solution C5914 (Sigma-aldrich, Saint Louis Missouri) (10 mL / flask) and 50 mL tube (TPP, Trasadingen Switzerland). The cells were centrifuged at 300 G for 5 minutes at 20 ° C. and the supernatant was discarded. Subsequently, the cells were washed with D-glucose-containing HBSS (without Mg ²⁺ and Ca ²⁺ ), centrifuged as described above, and resuspended in D-glucose-containing HBSS (without Mg ²⁺ and Ca ²⁺ ). The concentration of the cell suspension was determined.

B16 of the α-gal epitope. Conjugated radiolabels to BL6 melanoma cells and the normal α-Gal trisaccharide-carbamate (9) probe (labeled at the cyclic carbamate carbon) with D-glucose (Mg ²⁺ and Ca ²⁺ absent) HBSS (Gibco Dissolved in Invitrogen, Taastrup Denmark, and filtered through a sterile filter (0.2 μm Super® Acrodisc® 13, Gelman Sciences, Ann Arbor Michigan). Eight different concentrations of radiolabeled α-Gal trisaccharide-carbamate (9) (1 μg, 10 μg, 100 μg, 1 mg, 5 mg, 10 mg, 15 mg, and 20 mg), and six different concentrations of “Glycom conjugate” (10 μg, 100 μg, 1 mg, 5 mg, 10 mg, 15 mg, and 20 mg) were used. A total of 10 × 10 ⁶ B16. Twelve flasks (300 cm ² ) containing BL6 cells (TPP, Trasadingen Switzerland) for conjugation with radiolabeled trisaccharide probes and a total of 5 × 10 ⁶ B16. Eight flasks (150 cm ² ) containing BL6 cells (TPP, Trasingingen Switzerland) were prepared for conjugation with non-radioactive complexes. To the Glycom conjugate, add D-glucose-containing HBSS (without Mg ²⁺ and Ca ²⁺ ), make each large flask (300 cm ² ) to a final volume of 20 mL, and add D-glucose-containing HBSS (without Mg ²⁺ and Ca ²⁺ ). In addition, each medium flask (150 cm ² ) was brought to a final volume of 10 mL. The cells were incubated with “Glycom conjugate” at 37 ° C. for 1.5 hours. After conjugation, the supernatant from each flask was transferred to a different 50 mL tube (TPP, Trasadingen Switzerland). Cells were harvested with cell detachment solution (10 mL / large flask and 5 mL / medium flask) (Sigma-aldrich, Saint Louis Missouri), and the supernatant was transferred to a 50 mL tube (TPP, Trasdingen Switzerland). Cells were centrifuged at 300 G for 3 minutes at 20 ° C. and resuspended in 5 mL of PBS (without Mg ²⁺ and Ca ²⁺ ). This was done three times before determining cell concentration and viability by microscopic examination of trypan blue stained samples.

Cells bound to scintillation radiolabeled trisaccharide probe were centrifuged at 1000 G for 5 minutes at 20 ° C. The supernatant was discarded and the remaining pellet was dried. Subsequently, the pellet was dissolved in 450 μL of milli-Q water and 1.5 mL of scintillation solution was added. Standards for scintillation were prepared by adding 500 pCi, 0.5 pCi, and 0 pCi to the three control cells. Samples were stored at room temperature for 3 days until the cell suspension was transferred to scintillation vials and counted.

Flow cytometry analysis Cells conjugated with normal trisaccharide probes were analyzed by flow cytometry. 400 μL of each cell suspension containing 2 × 10 ⁵ cells was cultured with FITC-labeled GS1B4 at 4 ° C. for 15 minutes. The average fluorescence intensity of the cells stained with GS1B4 was measured by flow cytometry.

＊）α−ｇａｌ接合し、細胞を１回洗浄した後に決定した。 Table 18 Cells conjugated to normal complexes

*) Determined after α-gal conjugation and cell washing once.

＊）α−ｇａｌ接合し、細胞を４回洗浄した後に決定した。 Table 19 Cells conjugated with radiolabeled complex

*) Determined after α-gal conjugation and washing the cells 4 times.

Contrary to flow cytometry predictions, in any conjugation reaction, FITC-labeled GS1B4 and cells bound to cells did not exceed the above background. Additional FITC-labeled GS1B4 was added to the cells and the measurement was repeated. However, again no staining was seen and therefore flow cytometric analysis revealed that α-gal conjugation was not successful.

The results obtained with these cells were not included because of the suspicion of the number of cells conjugated to 1 mg of scintillation radiolabeled complex. In the remaining conjugation reactions, a positive correlation was seen between scintillation counts and complex doses. As described above, since α-gal junction could not be confirmed by flow cytometry, the relationship between the two quantitative methods could not be found.

＊）α−ｇａｌ接合し、細胞を４回洗浄した後に決定した。 Table 20 Cells conjugated with radiolabeled complex

*) Determined after α-gal conjugation and washing the cells 4 times.

Table 21 Epitope / cell as a function of conjugate dose

Part 2: Preparation of carbohydrate-containing cyclic carbamate

Lactosyl azide 12 (1.5 g) is dissolved in anhydrous DMF (40 mL) saturated with CO ₂ followed by addition of a solution of triphenylphosphine (1.1 eq) in anhydrous DMF (5 mL) over 20 min. Added (see FIG. 5). CO ₂ was bubbled through the mixture for 5 hours and the mixture was stirred for 8 hours. The produced white precipitate was filtered and washed with cold acetone to obtain the desired product 13 (1 g).

  Lactosyl azide 12 (1.8 g) was dissolved in anhydrous DCM (15 mL) followed by addition of triphenylphosphine (1.1 eq) to the mixture and stirred for 3 hours (see FIG. 6). Diethyl ether (50 mL) was added and the resulting white precipitate was filtered and washed with cold diethyl ether to give phosphinimine 16 (1.9 g).

CO ₂ was blown into a solution of the phosphinimine derivative (1.9 g) in anhydrous acetone (40 mL), and the mixture was stirred at room temperature for 6 hours. Subsequently, the produced white crystals were collected to obtain the target product 13.

At 0 ° C., triphosgene (1.1 equiv) was added to α-gal trisaccharide epitope 18 (1 g) in EtOAc and saturated NaHCO ₃ solution and the mixture was stirred vigorously for 30 min (see FIG. 7). Subsequently, the layers were separated and the organic layer was collected and concentrated. The residue was subjected to column chromatography to give product 19.

In the presence of DIPEA (1.3 eq), Z-CL (1.2 eq) was added to a DMF solution of lactosylamine 20 (1 g) and the mixture was stirred until conversion to compound 21 by TLC was complete ( (See FIG. 8). Subsequently, NaOMe (1.3 eq) was added and stirred at room temperature. The reaction mixture was neutralized with Amberlite IR 120 (H ⁺ ) and concentrated. The residue was subjected to column chromatography to obtain the intended product 13.

  Acyl chloride (4 eq) is added to a solution of glucosyl carbamate 22 (100 mg) in DCM (3 mL) and pyridine (4 mL) and the mixture is stirred overnight, followed by concentration of the mixture, chromatography of the residue on product 23 Was obtained (see FIG. 9).

A solution of trisaccharide-azide 24 (50 mg) in absolute DMF (0.3 mL) was added to a solution of PPh ₃ (31.4 mg) in absolute DMF (0.2 mL). The mixture was stirred for 2 hours under an inert atmosphere (see FIG. 10). Subsequently, 5 mL of concentrated H ₂ SO ₄ was added to (C13 labeled) BaCO ₃ (39.4 mg) and the generated CO ₂ was passed through the carbohydrate mixture. The mixture was stirred for 8 hours at room temperature. The mixture was subsequently stirred and triturated with DCM (50 mL) to isolate product 25 as a white solid.

Claims (18)

A method for preparing a carbohydrate-peptide complex comprising the cyclic carbamate (1)

(Wherein R ³ and R ⁴ are independently selected from the group consisting of hydroxyl, acetamide, and carbohydrate moieties; and R ⁵ is hydrogen, methyl, hydroxymethyl, acetamidomethyl, carboxyl, and X - _{(CH 2) r} - is selected from the group consisting _of wherein, X is a carbohydrate moiety, r is an integer selected from 0, 1, 2 and 3;)
Reacting with a peptide comprising at least one primary amino group.   The process of claim 1, wherein the reaction is carried out in a polar solvent such as water.   A process according to any preceding claim, wherein the reaction is carried out in the range of pH 6.5-10.5.   A method according to any preceding claim, wherein the peptide comprises at least 30 amino acid units.   The method according to any of the preceding claims, wherein the peptide is a cell surface or cell membrane bound protein.   15. A method according to any of the preceding claims, wherein the carbohydrate-peptide complex is a compound as defined in any of claims 7-14.   A carbohydrate-peptide complex obtained by the method according to claim 1. General formula 1:

(here,
R ¹ and R ² together with the lysine moiety present in between represent a peptide moiety;
R ³ and R ⁴ are independently selected from the group consisting of hydroxyl, acetamide, and carbohydrate moieties;
R ⁵ is selected from the group consisting of hydrogen, methyl, hydroxymethyl, acetamidomethyl, carboxyl, and X— (CH ₂ ) _r —, where X is a carbohydrate moiety and r is selected from 0 and 1 Is an integer;)
Carbohydrate-peptide conjugates comprising one or more moieties of: and pharmaceutically acceptable salts thereof. General formulas 1a, 1b, and 1c

(Where R ⁶ and R ⁷ are as defined in R ³ and R ⁴ of claim 1, R ⁹ is as defined in R ⁵ of claim 1, R ⁸ is hydroxyl, C Selected from the group consisting of _1-6 -alkoxy, C _2-20 -acyloxy, acetamide, and a carbohydrate moiety.)
The carbohydrate-peptide complex according to claim 8, comprising any one or more of the moieties. General formula 2:

(here,
R ¹¹ is an amino acid side chain;
R ² together with —NH—CHR ¹¹ —C (═O) — represents a peptide moiety with a total of at least 30 amino acids;
R ³ and R ⁴ are independently selected from the group consisting of hydroxyl, acetamide, and carbohydrate moieties;
R ⁵ is selected from the group consisting of hydrogen, methyl, hydroxymethyl, acetamidomethyl, carboxyl, and X— (CH ₂ ) _r —, where X is a carbohydrate moiety and r is 0, 1, 2, and An integer selected from 3;)
Carbohydrate-peptide conjugates comprising one or more moieties of: and pharmaceutically acceptable salts thereof. General formulas 2a, 2b, and 2c

(Where R ⁶ and R ⁷ are as defined in R ³ and R ⁴ of claim 3, R ⁹ is as defined in R ⁵ of claim 11, and R ⁸ is (Selected from the group consisting of hydroxyl, C _1-6 -alkoxy, C _2-20 -acyloxy, acetamide, and a carbohydrate moiety.)
The carbohydrate-peptide complex of claim 10 comprising one or more moieties.   The carbohydrate-peptide complex according to any one of claims 7 to 11, wherein the glycosyl moiety (saccharide moiety) represents a non-immunogenic carbohydrate.   The carbohydrate-peptide complex according to any one of claims 7 to 11, wherein the glycosyl moiety (saccharide moiety) represents an immunogenic carbohydrate.   The carbohydrate-peptide complex according to any one of claims 7 to 13, wherein the carbohydrate-peptide complex is a cell surface or cell membrane-bound protein.   A carbohydrate-peptide complex as defined in any of claims 7-14, wherein the total number of amino acid units of the peptide part is at least 30, in particular at least 100.   Carbohydrate-peptide complex as defined in any of claims 7-15 for use in medicine.   Use of the carbohydrate-peptide complex according to any one of claims 7 to 15 as a drug, diagnostic agent or in a diagnostic kit. (4a), (4b) and (4c)

(Wherein R ³ , R ⁴ , R ⁶ and R ⁷ are independently selected from the group consisting of hydroxyl, acetamide, and carbohydrate moieties;
R ⁵ and R ⁹ are independently selected from the group consisting of hydrogen, methyl, hydroxymethyl, acetamidomethyl, carboxyl, and X— (CH ₂ ) _r —, where X is a carbohydrate moiety and r is And an integer selected from 0, 1, 2, and 3; and R ⁸ and R ¹⁰ are independently composed of hydroxyl, C _1-6 -alkoxy, C _1-6 -acyloxy, acetamide, and a carbohydrate moiety. Selected from the group;)
Cyclic carbamates of oligosaccharides selected from: and pharmaceutically acceptable salts thereof.

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